Recognition by integrins of macromolecular ligands that contain Arg-Gly-Asp (RGD) sequences is a key event in many processes in the vasculature including hemostasis and angiogenesis, and is targeted by several therapeutics. This project will define the atomic basis for recognition and shape changes in integrin extracellular domains that regulate affinity for ligand and are linked by conformational transmission across the plasma membrane to cytosolic signaling pathways. The structure of the integrin a,]b|33 headpiece bound to the C-terminal y chain dodecapeptide of fibrinogen reveals binding of Lys-406 to the anb p-propeller pocket and unexpected binding of the Val-411 a-carboxyl group to the p3 MIDAS Mg21" and water-mediated coordination of the Asp-410 sidechain to the ADMIDAS Ca2^. A complex with the intact yC module will be sought to test the hypothesis that in general, the integrin P-propeller cap subdomain recognizes folded portions of macromolecular ligands, and an adjacent interface with the P I domain recognizes more mobile sequence motifs such as Arg-Gly-Asp and regulates affinity for ligand. Complex structures of the anbp3 and avp3 headpieces with yC module, its dodecapeptide, fibronectin FN3 modules 9 and 10, and perhaps other RGD-containing macromolecules will reveal 1) whether avp3 and anbp3 recognize the dodecapeptide in the same manner, 2) the generality of the two-site model for macromolecule binding described above, and 3) compare binding to these two sites of different ligands by the same integrin and different integrins to the same ligand. Structures of intact airbp3 will reveal whether the I-EGF1/I-EGF2 interface is spring loaded in an intact integrin, and reveal sites to which novel classes of antagonists that prevent integrin extension could bind. Structures of the domains that flank the integrin knees wouldreveal key changes in the a and Psubunit legs that occur upon extension. At least one novel RGD-bearing ligand will be characterized structurally. The RGD site in von Willebrand factor is in the VWC repeat which is structurally unknown, and is important in hemostasis in shear flow. In the large latent complex of TGF-P, an RGD in the latency-associated peptide is key to release of active TGF-P from storage in the extracellular matrix.